1. Field of the Invention
The present invention relates to processes for the purification of proteins. More specifically, methods for solubilizing and purifying proteins expressed in an insoluble form using low concentrations of chaotropic agents, such as guanidine salts, are provided. Also provided are methods for refolding proteins solubilized according to the present invention.
2. Related Art
The advent of recombinant DNA technology has brought about an entirely new generation of protein products. The ability to clone and express proteins in bacteria, yeast and mammalian cells has made it possible to produce therapeutics and industrially important proteins at economically feasible levels. However, the expression of high levels of recombinant proteins in Escherichia coli often results in the formation of inactive, denatured protein that accumulates in intracellular aggregates known as insoluble inclusion bodies. (Krueger et al., "Inclusion bodies from proteins produced at high levels in Escherichia coli," in Protein Folding, L. M. Gierasch and P. King (Eds), Am. Ass. Adv. Sci., 136-142 (1990); Marston, Biochem. J. 240:1-12 (1986); Mitraki, et al., Bio/Technol. 7: 800-807 (1989); Schein, Bio/Technol. 7:1141-1147 (1989); Taylor, etal., Bio/Technol. 4: 553-557 (1986)). Inclusion bodies are dense aggregates, which are 2-3 .mu.m in diameter and largely composed of recombinant protein, that can be separated from soluble bacterial proteins by low-speed centrifugation after cell lysis (Schoner, et al. Biotechnology 3:151-154 (1985)).
The recovery of recombinantly expressed protein in the form of inclusion bodies has presented a number of problems. First, although the inclusion bodies contain a large percentage of the recombinantly produced protein, additional contaminating proteins must be removed in order to isolate the protein of interest. Second, the proteins localized in inclusion bodies are in a form that is not biologically active, presumably due to incorrect folding.
Several methods have been developed to obtain active proteins from inclusion bodies. These strategies include the separation and purification of inclusion bodies from other cellular components, solubilization and reduction of the insoluble material, purification of solubilized proteins and ultimately renaturation of the proteins and generation of native disulfide bonds. The art teaches that concentrations of 6M or greater of chaotropic agents, such as guanidine hydrochloride, guanidine isothiocyanate or urea-are necessary for solubilization of the insoluble recombinant polypeptides from the inclusion bodies. See, for example, Vandenbroeck et al, Eur. J. Biochem. 215:481-486 (1993); Meagher et al., Biotech. Bioeng. 43:969-977 (1994); Yang et al., U.S. Pat. No. 4,705,848, issued Nov. 10, 1987; Weir et al., Biochem. J. 245:85-91 (1987); and Fischer, Biotech. Adv. 12:89-101 (1994).
Contrary to the teachings of the prior art, the inventors have discovered that low concentrations of guanidine salts can be used to solubilize biologically active (i.e., correctly folded) proteins and extract this population of the protein from a hetergenous protein mixture localized in inclusion bodies. The methods disclosed herein are particularly useful for the purification of chemokines.